title: Metabarcoding for NoV surveillance: Baseline Survey sample sequencing
description: Norovirus (NoV) specific metabarcoding was performed employing Illumina short-read sequencing. This amplicon-based approach takes advantage of the well-established primers used for NoV genotyping, that target specific, well-studied regions of the viral genome, namely portions of the genes encoding the RNA-dependent RNA polymerase (RdRp) and the capsid protein (VP1) (Imamura et al., 2017, Lun et al., 2018, Oshiki et al., 2018). In addition, primers that target a longer fragment (400-500 nt) comprising a part of RdRp and a part of VP1 gene sequences were also employed, thus allowing the typing of both genes in the same amplicon (dual typing). The PCR amplicons were tagged and indexed by an additional PCR, purified, quantified and mixed into a pool to perform sequencing on the Illumina Miseq platform. The raw reads, are thus, amplified genome fragment products that arise from different (Noro)viruses present in the sample. Raw reads were demultiplexed directly by the Illumina MiSeq sequencer. Reads quality was verified using FastQC (V.0.11.8 https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ Andrew, 2010) and MultiQC (V.1.9 https://multiqc.info/ Ewels et al., 2010) tools. Reads were assembled into clusters using the FROGS suite in a Galaxy environment (Escudié et al, 2018). The sequence of each cluster was genotyped using the Norovirus Typing Tool 2.0 to allow for the characterization of the diversity of the NoV genotypes. Samples come from two different sequencing sets - Baseline Survey (BLS) samples, and two synthetic Test Set samples (TSS1 and TSS2). The European Baseline Survey (BLS) of norovirus in oysters was undertaken by EFSA (2016-18). From November 2016 to October 2018, samples of BMS (flat oysters Ostrea edulis, Pacific oysters Crassostrea gigas, and Portuguese oysters Crassostrea angulata) were collected every two months from sampling locations in two points in the food chain (production areas and dispatch centers) in 13 oyster-producing countries in the EU. Only a sub-selection of 200 main (M) samples and 100 reserve (R) samples that were positive for NoV GI and/or GII were considered for analysis in this sequencing study, of which 212 were actually sequenced.